The nonmuscle myosin light chain kinase gene is very complex. It encodes for several alternatively spliced versions of myosin light chain kinase (MLCK) including a 205,000 Da version and a 150,000 version. The 205,000 Da version has an extended amino terminus. The 3' portion of the gene also encodes kinase related protein, also known as KRP or telokin, an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). We have previously determined that KRP binds to the junction between the light chain binding domain and the coiled-coil portion of unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky et al., (1993) J. Biol. Chem. 268, 16578-16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol KRP/1 mol myosin and an affinity of 5.5 uM. Previously, another group reported that caldesmon, another smooth muscle protein, was also able to stabilize smooth muscle myosin filaments. We have reinvestigated this question and find that caldesmon does not stabilize smooth muscle myosin filaments. We have begun to express the amino terminal portion of 205,000 Da MLCK in baculovirus and bacteria in order to characterize its properties. Preliminary experiments suggest that it is capable of binding to actin filaments.